DESCRIPTION: This project is concerned with the process of replication of UV- damaged DNA. Human cells are known to respond efficiently to UV radiation, as evidenced by activation of checkpoints that regulate cell- cycle progression, inhibition of replicon initiation and operation of excision repair. Still, they cannot avoid replicating DNA that contains photoproducts, since lesions deposited on replication units already initiated at the time of UV exposure are likely to be encountered by the replication machinery. This cis-acting effect of DNA damage on replication apparati has been accepted as the basis for the observed UV- induced inhibition of DNA strand growth. Blockage of fork displacement and Okazaki fragments by template lesions are thought to account for the appearance of daughter-strand gaps in nascent DNA and the attendant reduction in its molecular weight distribution. This UV effect is exacerbated in xeroderma pigmentosum variant (XPV) cells and it is predictive of their enhanced sensitivity to mutagenesis by UV. The applicant originally proposed that the abnormal response of XPV cells to UV was due to the absence of a gene product that in normal cells contributes to the error-free translesion synthesis of pyrimidine dimers. Thus, the principal investigator investigated whether the activity of this gene product could be detected in extracts of human cells when UV-irradiated plasmid DNA was replicated in vitro. A higher inhibition of plasmid DNA replication was observed with XPV extracts, compared to those from other human cells, but not the expected hypermutability. In this renewal application it is proposed to investigate the alternative hypothesis that DNA damage activates transacting regulators that promote the slowing down and/or arrest of replication forks, even before they themselves are blocked by DNA lesions. In this scenario, the XPV phenotype is explained by an imbalanced UV-dependent response that leads to a more severe inhibition of DNA strand growth than warranted by the level of DNA damage. This hypothesis will be tested by quantifying the in vivo replication of irradiated plasmid DNA in XPV and normal cells (Aim 1), and then determining whether UV lesions in one plasmid can lead to inhibition of replication of an undamaged one (Aim 2). If a down-stream effect of this response to UV is to modify the replication machinery, so as to favor completion of DNA replication in the damaged cells at the expense of decreasing replication fidelity, the consequence will be increased mutagenesis. With a dual shuttle vector system we will establish whether this mutagenic response is specific to UV (Aim 2). Another facet of this paradigm will be tested by determining the frequencies of UV-induced mutations in vitro (Aim 3), when extracts are prepared at different times following irradiation of XPV and normal cells with low fluences of UV. In Aim 4 the principal investigator will determine rates of bypass of the T[c,s]T dimer and its mutagenic potential, with extracts prepared under conditions defined in previous aims. These studies will provide novel information on the response of human cells to UV damage and its relevance to mutagenesis and carcinogenesis by solar radiation.